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Laboratorium voorMicrobiologie, universiteit Gent, Belgium We designed a panel of four 16s rRNA-targeted oligonucleotide probes specific for bacteria of the phylum cytophaga-flavobacter-bacteroides (CFB). Probes CF319a and CF319b are targeted to members O f the flavobacteria-cytophaga group and the genus Porphyromonas, whereas probe BAC303 has a target region characteristic for the genera Prevote//a and Bacteroides within the bacteroides group. The probe FFE8b was developed for species-specif ic hybridizations with Flavobacterium ferrugineum. All probes were designed by computer-assisted sequence analysis and compared to all currently accessible 16s and 235 rRNA sequences. The oligonucleotides were further evaluated by whole-cell and non-radioactive dot-blot hybridization against reference strains of the CFB phylum and other major lineages of Bacteria. The newly developed probes were used together with other higher-order probes t o analyse the structure and community composition in complex environments. In activated sludge samples, members of the f lavobacteria-cytophaga group were revealed by in situ hybridization as important constituents of sludge flocs and characteristic colonizers of filamentous bacteria. By application of fluorescent probe BAC303, members of the genera Bacteroides and Prewotella could be visualized without prior cultivation as an important part of the human faecal microf lora.

Emerging pathogens in drinking water have become increasingly important during the decade. These include newly-recognized pathogens from fecal sources such as Cryptosporidium parvum, Campylobacter spp., and rotavirus, as well as pathogens that are able to grow in water distribution systems, like Legionella spp., mycobacteria, and aeromonads. To perform a risk analysis for the pathogens in drinking water, it is necessary to understand the ecology of these organisms. The ecology of the drinking-water distribution system has to be evaluated in detail, especially the diversity and physiological properties of water bacteria. The interactions between water bacteria and (potential) pathogens in such diverse habitats as free water and biofilms are essential for the survival or growth of hygienically relevant organisms in drinking water. Results of epidemiological studies together with ecological data are the basis for effective resource protection, water treatment, and risk assessment.

Enhanced biological phosphate removal in an anaerobic-aerobic activated sludge system has generally been ascribed to members of the genus Acinetobacter. A genus-specific 16S rRNA-targeted oligonucleotide probe was developed to investigate the role ofAcinetobacter spp. in situ. Nonisotopic dot blot hybridization to 66 reference strains, including the seven described Acinetobacter spp., demonstrated the expected probe specificity. Fluorescent derivatives were used for in situ monitoring of Acinetobacter spp. in the anaerobic and aerobic compartments of a sewage treatment plant with enhanced biological phosphate removal. Microbial community structures were further analyzed with oligonucleotide probes specific for the alpha, beta, or gamma subclasses of the class Proteobacteria, for the Cytophaga-Flavobacterium cluster, for gram-positive bacteria with a high G+C DNA content, and for all bacteria. Total cell counts were determined by 4',6-diamidino-2-phenylindole staining. In both the anaerobic and the aerobic basins, the activated sludge samples were dominated by members of the class Proteobacteria belonging to the beta subclass and by gram-positive bacteria with a high G+C DNA content. Acinetobacter spp. constituted less than 10% of all bacteria. For both basins, the microbial community structures determined with molecular techniques were compared with the compositions of the heterotrophic saprophytic microbiota determined with agar plating techniques. Isolates on nutrient-rich medium were classified by whole-cell hybridization with rRNA-targeted probes and fatty acid analysis. Cultivation on nutrient-rich medium favored the growth of members of the gamma subclass of Proteobacteria and selected against the growth of members of the beta subclass of Proteobacteria and gram-positive bacteria with a high G+C DNA content; 33% of the cultured bacteria from the anaerobic basin and 32% from the aeration basin were identified as Acinetobacter spp. The addition of small amounts of iron salts for chemical phosphate precipitation had no influence on the constitution of the microbial consortia. Enrichment of the return sludge with 20 mg of acetic acid per liter for 3 days significantly increased the relative abundance of gram-positive bacteria with a high G+C DNA content but had no effect on the numbers of Acinetobacter spp. The dominance of gram-positive bacteria with a high G+C DNA content and the presence of polyphosphate inclusions in these bacteria indicate that they may play a major role in biological phosphate removal.

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> Abstract The phylogenetic composition, three-dimensional structure and dynamics of bacterial communities in river biofilms generated in a rotating annular reactor system were studied by fluorescent in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Biofilms grew on independently removable polycarbonate slides exposed in the reactor system with natural river water as inoculum and sole nutrient and carbon source. The microbial biofilm community developed from attached single cells and distinct microcolonies via a more confluent structure characterized by various filamentous bacteria to a mature biofilm rich in polymeric material with fewer cells on a per-area basis after 56 days. During the different stages of biofilm development, characteristic microcolonies and cell morphotypes could be identified as typical features of the investigated lotic biofilms. In situ analysis using a comprehensive suite of rRNA-targeted probes visualized individual cells within the alpha-, beta-, and gamma-Proteobacteria as well as the Cytophaga-Flavobacterium group as major parts of the attached community. The relative abundance of these major groups was determined by using digital image analysis to measure specific cell numbers as well as specific cell area after in situ probing. Within the lotic biofilm community, 87% of the whole bacterial cell area and 79% of the total cell counts hybridized with a Bacteria specific probe. During initial biofilm development, beta-Proteobacteria dominated the bacterial population. This was followed by a rapid increase of alpha-Proteobacteria and bacteria affiliated to the Cytophaga-Flavobacterium group. In mature biofilms, alpha-Proteobacteria and Cytophaga-Flavobacteria continued to be the prevalent bacterial groups. Beta-Proteobacteria constituted the morphologically most diverse group within the biofilm communities, and more narrow phylogenetic staining revealed the importance of distinct phylotypes within the beta1-Proteobacteria for the composition of the microbial community. The presence of sulfate-reducing bacteria affiliated to the Desulfovibrionaceae and Desulfobacteriaceae confirmed the range of metabolic potential within the lotic biofilms.http://link.springer-ny.com/link/service/journals/00248/bibs/37n4p225.html

Biofilms, accumulations of microorganisms at interfaces, have been described for every aqueous system supporting life. The structure of these microbial communities ranges from monolayers of scattered single cells to thick, mucous structures of macroscopic dimensions (microbial mats; algal-microbial associations; trickling filter biofilms). During recent years the structure of biofilms from many different environments has been documented and evaluated by use of a broad variety of microscopic, physico-chemical and molecular biological techniques, revealing a generally complex 3D structure. Parallel to these investigations more and more complex mathematical models and simulations were developed to explain the development, structures, and interactions of biofilms. The forces determining the spatial structure of biofilms, including microcolonies, extracellular polymeric substances (EPS), and channels, are still the subject of controversy. To achieve conclusive explanations for the structures observed in biofilms the cooperation of both fields of investigation, modelling and experimental research, is necessary. The expanding field of molecular techniques not only allows more and more detailed documentation of the spatial distribution of species, but also of functional activities of single cells in their biofilm environment. These new methods will certainly reveal new insights in the mechanisms involved in the developmental processes involved in the formation and behavior of biofilms.

It is recognized that microorganisms inhabiting natural sediments significantly mediate the erosive response of the bed (“ecosystem engineers”) through the secretion of naturally adhesive organic material (EPS: extracellular polymeric substances). However, little is known about the individual engineering capability of the main biofilm components (heterotrophic bacteria and autotrophic microalgae) in terms of their individual contribution to the EPS pool and their relative functional contribution to substratum stabilisation. This paper investigates the engineering effects on a non-cohesive test bed as the surface was colonised by natural benthic assemblages (prokaryotic, eukaryotic and mixed cultures) of bacteria and microalgae. MagPI (Magnetic Particle Induction) and CSM (Cohesive Strength Meter) respectively determined the adhesive capacity and the cohesive strength of the culture surface. Stabilisation was significantly higher for the bacterial assemblages (up to a factor of 2) than for axenic microalgal assemblages. The EPS concentration and the EPS composition (carbohydrates and proteins) were both important in determining stabilisation. The peak of engineering effect was significantly greater in the mixed assemblage as compared to the bacterial (x 1.2) and axenic diatom (x 1.7) cultures. The possibility of synergistic effects between the bacterial and algal cultures in terms of stability was examined and rejected although the concentration of EPS did show a synergistic elevation in mixed culture. The rapid development and overall stabilisation potential of the various assemblages was impressive (x 7.5 and ×9.5, for MagPI and CSM, respectively, as compared to controls). We confirmed the important role of heterotrophic bacteria in “biostabilisation” and highlighted the interactions between autotrophic and heterotrophic biofilm consortia. This information contributes to the conceptual understanding of the microbial sediment engineering that represents an important ecosystem function and service in aquatic habitats.